Human fcr blocking

Blood samples were collected in a BD vacutainer K3EDTA. Samples were incubated with human FcR Blocking (Miltenyi; Cologne, Germany) for 10 min at room temperature and incubated for 20 min at 4°C with directly conjugated antibodies diluted in phosphate-buffered saline with 1% fetal bovine serum. Samples were incubated with Quicklisis buffer (Cytognos; Salamanca, Spain) to lyse erythrocytes. The acquisition and analysis were conducted in a MacsQuant10 Flow Cytometer (Miltenyi). The antibodies employed and the flow cytometry gating strategy of the immune subpopulation analysis in dog-derived samples have been previously described.6 (link)

T cells were stained with anti-hCD45RA-PE (1:400) or anti-hCD45RA.FITC (1:400), anti-hCD62L-PE-Cy7 (1:400), and anti-hCD3-PerCP-Cy5 (1:400) (all from eBioscience-Invitrogen) during 30 min on ice. CAR expression was measured indirectly by the surface expression of truncated EGFR with anti-hEGFR-PE (Biolegend, 1:100). Then, cells were washed with PBS at 400 g for 5 min. Fluorescence was immediately acquired in a FACSCantoII cytometer, and data were analyzed with FlowJo v10 (TreeStart).
In the case of humanized murine samples, cells were washed with cold PBS + 2% BSA + 2 mM EDTA (“FACS buffer”) and incubated with anti-murine CD16/CD32 (Thermo Fisher, 1:100), human FcR blocking (Miltenyi, 1:00), and 5% of mouse serum for 20 min on ice. After one wash with FACs buffer, cells were stained with anti-human CD3-PerCP-Cy5.5 (1:200) for Jurkat-mice model or with anti-human CD19-APC (1:200, eBiosciences) for Namalwa mice model during 30 min on ice and dark for 30 min. Cells were fixed with fresh PFA (2%) prior to acquisition.

Fluorescence-activated cell sorting (FACS) for CD19 antigen expression analysis was performed on fresh low passage lymphoma cell lines. Cells were washed with ice-cold FACS buffer (PBS + 0.5% BSA) and divided 1×10⁶ cells/tube. A pretreatment with human FcR blocking (Miltenyi Biotec Inc., Auburn, CA, USA) was performed according to the manufacturer’s instructions. CD19-PE (5 μL; 1μg) or control isotype was incubated with cells at 4°C for 30 minutes. Cells were washed twice and re-suspended in FACS buffer. Flow-cytometry analysis was carried out with a FACS Canto II instrument (BD Biosciences). Median Fluorescence intensity (MFI) of each sample was determined using FacsDiva v.8.0.1 software (BD Biosciences, Allschwil, Switzerland). Unstained cells and cells stained with isotype control antibody were used as controls. RNA expression levels, obtained with the HumanHT-12 v.4 Expression BeadChip (Illumina, San Diego, CA, USA) and with the HTG EdgeSeq Oncology Biomarker panel (HTG Molecular Diagnostics Inc., Tucson, AZ, USA) were published previously.^{26 (link)} For leukemia cell lines, the median number of CD19 antibody binding sites (ABC) was determined as previously described.^{24 (link)} Pairwise correlations were assessed using Stata/SE 12.1 for Mac. P<0.05 was considered statistically significant.